CN1637133A - Banana endophyte and its use - Google Patents
Banana endophyte and its use Download PDFInfo
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- CN1637133A CN1637133A CN 200410052595 CN200410052595A CN1637133A CN 1637133 A CN1637133 A CN 1637133A CN 200410052595 CN200410052595 CN 200410052595 CN 200410052595 A CN200410052595 A CN 200410052595A CN 1637133 A CN1637133 A CN 1637133A
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Abstract
The present invention relates to banana endogenous streptomycete, its separation and culture method and its use in promoting banana growth and preventing and treating banana fusarium blight. The banana endogenous streptomycete CCTCC M203070 may be obtained from banana plant in banana blight area through separation and culture. Experiment shows that the streptomycete CCTCC M203070 has the functions of promoting banana seedling growth and raising the resistance to fusarium blight, so that it may be used in promoting banana growth and preventing and treating banana fusarium blight.
Description
Technical field
The present invention relates to a kind of plant endogenesis actinomycetes---banana endogenetic streptomycete and isolation cultivation method thereof and the purposes that is used for short length of banana and the control of banana sickle spore bacterium (fusarium oxysporum bacterium Fusarium oxysporum) blight.
Background technology
Endophyte of plant is to separate from the process plant tissue of surface sterilization or to obtain from the juice of inside plants, and apparent the going up of plant do not had the microorganism that harm does not promptly cause obvious illness.Endophyte of plant comprises reciprocal sharp altogether (mutualistic) and neutral (neutral) endosymbiontic microorganism.At present from the various organs (root, stem, leaf, flower, fruit, seed) of a lot of plants, be separated to endophyte.They have many reported in literature at the intravital ubiquity of plant, abundant species diversity and functional diversity.Endophyte interacts with plant through secular, forms complicated relation between them.Have now found that endophyte has the effect of the following aspects to host plant:
1) promotes the host plant growth, improve host's productivity, can strengthen the host to resistance sick, worm, thereby show significance aspect agro-ecology control, the biological control;
2) strengthen the resistance of host plant to environment-stress.As resistance to arid, global warning, saline and alkaline, heavy metal and other toxic substance pollution etc.;
3) endophyte produces new biologically active substance.Endophyte of plant is as a kind of microorganism of special habitats, in plant materials, produce rich and varied, have a multiple bioactive secondary metabolite, people have found from endophyte and have manyly had various bioactive novel substances and demonstrate important use at pharmaceutical sector, aspect agriculture and be worth at present.
At present, few about the actinomycetic research of interior life in the world, more do not carry out the test report that the banana endogenetic streptomycete is used for resisting banana vascular wilt.
Summary of the invention
The object of the present invention is to provide a kind of purposes that can prevent and treat the banana endophyte and the isolation cultivation method thereof of banana sickle spore bacterium blight and be used for short length of banana and the control of banana sickle spore bacterium blight.
Banana endophyte of the present invention is the strain actinomycetes that the contriver is separated to from banana plant, shows that through traditional method evaluation and Molecular Identification it belongs to streptomycete, with its called after streptomycete S96 (Streptomyces roseogriseolus S96).
Banana endophyte of the present invention---streptomycete S96 (Streptomyces roseogriseolus S96) is deposited in Chinese typical culture collection center (CCTCC) on September 22nd, 2003, and deposit number is CCTCC NO.M203070.
Banana endophyte of the present invention---streptomycete S96 (being designated hereinafter simply as streptomycete CCTCC M203070) has following biological property:
1. colony morphology characteristic: well-grown on the S substratum, the actinomycetes colony characteristics: mycelia is tight, positive grey, the back side is the melon pulp pink colour, produces fallen or falling flowers pale pink soluble pigment, and spore connects into the straight chain shape, and spore count is greater than 15.
2. morphological features: cell is thread, and hyphal diameter is about 0.3-0.7 μ M, Gram-positive.
3. major physiological biochemical character: can well growth on actinomycetes substratum S, growth temperature 10-38 ℃, growth pH5-9; There is obvious inhibition active to the fusarium oxysporum bacterium, produces amylase, zytase, dextranase and cellulase.
4. genetics feature: the 16S rDNA portion gene sequence (709bp) of this streptomycete CCTCC M203070 is submitted to the Genbank geneseq database of American National biotechnology information center (NCBI), and accession number is AJ519793.According to " classification of actinomycetes and evaluation " (Yan Xunchu, Science Press, 1992), streptomycete CCTCC M203070 of the present invention is on morphological specificity, and is the most approaching with the rose light gray streptomycete (Streptomycesroseogriseolus) of domestic name; The 16S rDNA sequential analysis of this streptomycete CCTCC M203070 shows, is 99% to the similarity of the most similar bacterial strain Streptomyces griseorubiginosus (AJ399488) that writes down in the Genbank international data center.Therefore, streptomycete CCTCC M203070 should belong to streptomyces.
Streptomycete CCTCC M203070 of the present invention can obtain from the banana plant separation and Culture by the following method:
(1) root of the banana plant that will gather in the banana blight epidemic-stricken area, stem are fritter or/and leaf after surface sterilization is handled, is cut, and are inoculated in the S culture medium flat plate of S or improvement, cultivate 5～7 days down in 24～30 ℃;
(2) the small-sized mycelia of picking actinomycetes feature bacterium colony closely in same medium plate streaking purifies and separates, places 24～30 ℃ to cultivate 5～7 days;
(3) the pure bacterial strain of gained and pathogenic bacteria fusarium oxysporum bacterium are done dull and stereotyped face-off test, therefrom select bacterial strain pathogenic bacteria tool resistance, further verify its short long and anti-disease activity by the banana seedlings inoculation test after, identify also preservation bacterial classification.
The contriver has been developed a cover culture technique at this streptomycete CCTCC M203070, and can be this bacterium tieback again to tissue culture seedlings of bananas, promoted banana seedlings growth, strengthened the resistance of banana to fusarium wilt.
The producing method for seed of streptomycete CCTCC M203070 of the present invention, its step comprises:
(1) streptomycete CCTCC M203070 pure strain is inoculated in S or improvement S medium slant, it is plentiful to be cultured to spore at 28 ℃, as the inclined-plane seed;
(2) the inclined-plane seed is connected in the seed culture medium, under 25～29 ℃, cultivated 30-50 hour, as secondary seed solution with 150～200r/m speed oscillation; Described seed culture medium is the S substratum of S or improvement, pH7.3;
(3) insert secondary seed solution at solid-state production of hybrid seeds substratum by 2～4% (volume/weight), 28 ℃ of heat insulating culture are plentiful to spore; The results spore is as bacterial classification; Described production of hybrid seeds medium component consists of: Semen Maydis powder 150g, glucose or sucrose 100g, soya bean grouts powder 150g, peptone 10g, yeast extract paste or yeast powder 10g, lime carbonate 50g and water 300ml.
S medium component composition of the present invention is generally: glucose 1.0g, peptone 4.0g, phosphoric acid hydrogen dimethylamino potassium 0.5g, sal epsom 0.1g, calcium chloride 0.1g, (trace elements) such as ironic citrates of trace, agar 2.0g, seawater 100ml, pH7.0～7.3; The S substratum of described improvement normally replaces peptone with saltpetre 1.0g on the S medium base.
Banana endophyte of the present invention---streptomycete CCTCC M203070 can not only promote the banana growth, and can be used for preventing and treating banana sickle spore bacterium (fusarium oxysporum bacterium Fusarium oxysporum) blight.This streptomycete CCTCC M203070 has all shown the activity of antagonism pathogenic bacteria fusarium oxysporum bacterium Cuba specialized form through internal and external test.Test on banana tissue extract substratum shows that the anti-mycotic activity of this bacterium may be relevant with the effective iron in the contention banana tissue.Evidence, use this banana endophyte inoculation banana seedlings after, plant growth condition improves, disease resistance improves.
When banana endophyte of the present invention is used to promote banana growth and control banana sickle spore bacterium blight, only needing this banana endophyte---streptomycete CCTCC M20307 is inoculated in banana seedlings, can obtain to promote the banana seedlings growth and improve its effect to banana sickle spore bacterium blight disease resistance.
Embodiment
Embodiment 1:
One of isolation cultivation method of streptomycete CCTCC M203070:
1. gather the root sample of the healthy banana plant in banana blight epidemic-stricken area, fully flushing, 70% Ethanol Treatment 5 minutes, clorox (0.9% available chlorine) solution-treated 20 minutes, sterilized water fully washes;
2. the sample of handling well is shredded, insert in the S flat board;
3. this flat board places 26 ℃ to cultivate 5 days;
4. the small-sized mycelia of picking actinomycetes feature bacterium colony closely in same medium plate streaking purifies and separates, places 24～30 ℃ to cultivate 5 days, and picking list bacterium colony is connected to the same medium inclined-plane and preserves.
5. the pure bacterial strain of gained and pathogenic bacteria fusarium oxysporum bacterium are done dull and stereotyped face-off test, therefrom select bacterial strain pathogenic bacteria tool resistance, further verify its short long and anti-disease activity by the banana seedlings inoculation test after, identify also preservation bacterial classification.
Embodiment 2:
Two of the isolation cultivation method of streptomycete CCTCC M203070:
1, gather the leaf sample of the healthy banana plant in banana blight epidemic-stricken area, fully flushing, 36% formaldehyde treated 30 seconds, clorox (0.9% available chlorine) solution-treated 20 minutes, sterilized water fully washes;
2, the sample of handling well is shredded, insert in the S flat board of improvement;
3, this flat board places 26 ℃ to cultivate 7 days;
4, the small-sized mycelia of picking actinomycetes feature bacterium colony closely in same medium plate streaking purifies and separates, places 24～30 ℃ to cultivate 5 days, and picking list bacterium colony is connected to the same medium inclined-plane and preserves.
5, the pure bacterial strain of gained and pathogenic bacteria fusarium oxysporum bacterium are done dull and stereotyped face-off test, therefrom select bacterial strain pathogenic bacteria tool resistance, further verify its short long and anti-disease activity by the banana seedlings inoculation test after, identify also preservation bacterial classification.
Embodiment 3:
One of cultivation producing method for seed of streptomycete CCTCC M203070:
1. the purebred S of the connecing medium slant of streptomycete CCTCC M203070 that embodiment 1 or 2 is obtained;
2.28 ℃ being cultured to spore covers with;
3. switching same medium flat board is cultured to spore in 28 ℃ and covers with; Spore can directly apply to inoculation banana or enlarged culturing.
Embodiment 4:
Two of the cultivation producing method for seed of streptomycete CCTCC M203070:
1. the purebred S of the connecing medium slant of streptomycete CCTCC M203070 that embodiment 1 or 2 is obtained, 28 ℃ are cultured to spore and cover with;
2. the inclined-plane seed is connected to and contains in the 200ml of 500ml triangular flask seed culture medium, cultivated 30 hours with the 200r/m speed oscillation, be secondary seed solution in 28 ℃;
3. adorn the solid-state production of hybrid seeds substratum of about 1/5th sterilizations in the triangular flask, insert secondary seed solution by 2～4% (V/W), be mixed, 28 ℃ of heat insulating culture to spores cover with, and the spore of acquisition can be used for inoculating banana.
Described production of hybrid seeds substratum is: Semen Maydis powder 150g, glucose (or sucrose) 100g, soya bean grouts powder 150g, peptone 10g, yeast extract paste or yeast powder 10g, lime carbonate 50g, the about 300ml of water.
Embodiment 5:
According to the method for embodiment 4, " the adorning the solid-state production of hybrid seeds substratum of about 1/5th sterilizations in the triangular flask " in its step 3 changes " the sterilization of solid production of hybrid seeds substratum of about three cm thicks of dress one deck in the porcelain dish " into, and other step is identical with embodiment 4 with condition.
Embodiment 6:
Streptomycete CCTCC M203070 is applied to the test of resisting banana vascular wilt pathogenic bacteria:
1. the purebred S medium slant that connects of streptomycete CCTCC M203070,28 ℃ are cultured to spore and cover with;
2.. wash inclined-plane endophyte spore, be inoculated in the end of banana caulo stick (about 3 centimetres) A, and be cultured to spore and on false stem, grow and form visible bacterium colony with sterilized water;
3.. and then pathogenic fungi fusarium oxysporum bacterium is inoculated in the end of the nothing inoculation streptomycete CCTCC M203070 of the end of banana caulo stick B and false stem piece A, cultivated 5 days for 28 ℃;
4.. use the growth of pathogenic fungi on the microscopic examination banana caulo stick after with the naked eye direct viewing dyes with employing lactic acid phenol indigo plant, the influence that the judgement endophyte is grown to pathomycete;
5.. result: the false stem piece B of chain link mould CCTCC M203070 does not cover with the pathomycete mycelia very soon, and false stem piece A only has and invaded by the pathomycete mycelia in two minutes one, and an end that has met streptomycete CCTCC M203070 be can't see the pathomycete mycelia; After the blue dyeing of lactic acid phenol, microscopic examination is also found to organize seldom pathomycete mycelial growth near the stem of streptomycete CCTCC M203070.
Embodiment 7:
Streptomycete CCTCC M203070 is applied to the test of resisting banana vascular wilt:
1. the purebred S medium slant that connects of streptomycete CCTCC M203070,28 ℃ are cultured to spore and cover with;
2. the inclined-plane seed is connected to and contains in the 200ml of 500ml triangular flask seed culture medium, cultivated about 30 hours with the 200r/m speed oscillation, be secondary seed solution in 28 ℃;
3. adorn the solid-state production of hybrid seeds substratum of about 1/5th sterilizations in the triangular flask, by 2～4% access secondary seed solution, be mixed, 28 ℃ of heat insulating culture to spores cover with;
4. with 20 strains of above-mentioned streptomycete CCTCC M203070 inoculum inoculation tissue culture seedlings of bananas, tissue cultured seedling moves to the greenhouse after being transferred to flowerpot, and the banana seedlings upgrowth situation, plant height and the blade face area that have as seen met streptomycete CCTCC M203070 after one month all obviously are better than control group;
5. banana seedlings was transplanted back 45 days, carry out the pathogenic Fusarium oxysporum of artificial challenge, after two months, Huang Yehe to occur not withered gradually for control group 100% banana seedlings of chain link mould CCTCC M203070, the test group banana seedlings of having inoculated streptomycete CCTCC M203070 grows fine, and 100% no blight symptom occurs;
Embodiment 8:
Streptomycete CCTCC M203070 is to the expanding test of banana growth effect:
1. the purebred S medium slant that connects of streptomycete CCTCC M203070,28 ℃ are cultured to spore and cover with;
1. the inclined-plane seed is connected to and contains in the 200ml of 500ml triangular flask seed culture medium, cultivated 30 hours with the 200r/m speed oscillation, be secondary seed solution in 28 ℃;
2. adorn the solid-state production of hybrid seeds substratum of about 1/5th sterilizations in the triangular flask, by 2～4% access secondary seed solution, be mixed, 28 ℃ of heat insulating culture to spores cover with;
3. with 100 strains of above-mentioned streptomycete CCTCC M203070 inoculum inoculation tissue culture seedlings of bananas, tissue cultured seedling migrates to the nursery then, as seen the banana seedlings upgrowth situation of inoculating streptomycete CCTCC M203070 after two months obviously is better than control group, and plant height and blade face area increase about 1/3rd than control group.
Claims (9)
1. banana endophyte, it is characterized in that: this bacterium is streptomycete CCTCC M203070.
2. according to the described banana endophyte of claim 1, it is characterized in that this bacterium obtains from the banana plant separation and Culture.
3. according to the described banana endophyte of claim 2, it is characterized in that this bacterium obtains from the banana plant separation and Culture by the following method:
(1) root of the banana plant that will gather in the banana blight epidemic-stricken area, stem are fritter or/and leaf after surface sterilization is handled, is cut, and are inoculated in the S culture medium flat plate of S or improvement, cultivate 5～7 days down in 24～30 ℃;
(2) the small-sized mycelia of picking actinomycetes feature bacterium colony closely in same medium plate streaking purifies and separates, places 24～30 ℃ to cultivate 5～7 days;
(3) the pure bacterial strain of gained and pathogenic bacteria fusarium oxysporum bacterium are done dull and stereotyped face-off test, therefrom select bacterial strain pathogenic bacteria tool resistance, further verify its short long and anti-disease activity by the banana seedlings inoculation test after, identify also preservation bacterial classification.
4. according to the described banana endophyte of claim 3, it is characterized in that: the S substratum of described improvement is to replace peptone with 1.0g saltpetre on the S medium base.
5. the producing method for seed of the described banana endophyte of claim 1, its step comprises:
(1) streptomycete CCTCC M203070 pure strain is inoculated in S or improvement S medium slant, it is plentiful to be cultured to spore at 28 ℃, as the inclined-plane seed; The S substratum of described improvement is to replace peptone with saltpetre 1.0g on the S medium base;
(2) the inclined-plane seed is connected in the seed culture medium, under 25～29 ℃, cultivated 30-50 hour, as secondary seed solution with 150～200r/m speed oscillation; Described seed culture medium is the S substratum of S or improvement, pH7.3;
(3) insert secondary seed solution at solid-state production of hybrid seeds substratum by 2～4% volume/weight ratio, 28 ℃ of heat insulating culture are plentiful to spore; The results spore is as bacterial classification; Described production of hybrid seeds medium component consists of: Semen Maydis powder 150g, glucose or sucrose 100g, soya bean grouts powder 150g, peptone 10g, yeast extract paste or yeast powder 10g, lime carbonate 50g and water 300ml.
6. the described banana endophyte of claim 1 is used to prevent and treat the purposes of banana sickle spore bacterium blight.
7. according to the described purposes of claim 6, it is characterized in that: utilize streptomycete CCTCC M203070 to be inoculated in banana seedlings, obtain the banana plant that sickle spore bacterium blight is had disease resistance.
8. the described banana endophyte of claim 1 is used for the short long purposes of banana.
9. according to the described purposes of claim 8, it is characterized in that: utilize streptomycete CCTCC M203070 to be inoculated in banana seedlings, promote the banana seedlings growth.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100363058C (en) * | 2006-03-21 | 2008-01-23 | 浙江理工大学 | Sterilizing method for separating plant endogenous fungus |
| CN102206593A (en) * | 2011-03-15 | 2011-10-05 | 海南大学 | Streptomyces aureoverticillatus strain and its application |
| CN102329755A (en) * | 2011-09-28 | 2012-01-25 | 中国热带农业科学院海口实验站 | Nonabsorbent streptomyces ahygroscopicus strain BWL58 for preventing and treating banana vascular wilt and application thereof |
| CN103392414A (en) * | 2013-07-01 | 2013-11-20 | 句容根生药材有限公司 | Salvia miltiorrhiza seed disease-resistant treatment method |
| CN104894018A (en) * | 2015-05-27 | 2015-09-09 | 中国热带农业科学院热带生物技术研究所 | Luna Lin Haresii streptomycete and application thereof |
| CN106244491A (en) * | 2016-08-29 | 2016-12-21 | 佛山市艳晖生物科技有限公司 | A kind of banana stem leaves residuum decomposing microbial inoculum suppressing droop and preparation method thereof |
| CN107904196A (en) * | 2017-12-26 | 2018-04-13 | 中国热带农业科学院热带生物技术研究所 | A kind of Yan Shi streptomycetes and its application |
| CN108977381A (en) * | 2018-08-07 | 2018-12-11 | 广西壮族自治区农业科学院 | Raw bacillus amyloliquefaciens and its application in a kind of banana |
| CN110066755A (en) * | 2019-05-20 | 2019-07-30 | 江苏师范大学 | One plant of fructus arctii endogenetic streptomycete, the microbial bacterial agent containing the endophyte and application |
-
2004
- 2004-12-09 CN CNB2004100525951A patent/CN100360660C/en not_active Expired - Fee Related
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100363058C (en) * | 2006-03-21 | 2008-01-23 | 浙江理工大学 | Sterilizing method for separating plant endogenous fungus |
| CN102206593A (en) * | 2011-03-15 | 2011-10-05 | 海南大学 | Streptomyces aureoverticillatus strain and its application |
| CN102206593B (en) * | 2011-03-15 | 2012-11-28 | 海南大学 | Streptomyces aureoverticillatus strain and its application |
| CN102329755A (en) * | 2011-09-28 | 2012-01-25 | 中国热带农业科学院海口实验站 | Nonabsorbent streptomyces ahygroscopicus strain BWL58 for preventing and treating banana vascular wilt and application thereof |
| CN102329755B (en) * | 2011-09-28 | 2013-07-10 | 中国热带农业科学院海口实验站 | Nonabsorbent streptomyces ahygroscopicus strain BWL58 for preventing and treating banana vascular wilt and application thereof |
| CN103392414A (en) * | 2013-07-01 | 2013-11-20 | 句容根生药材有限公司 | Salvia miltiorrhiza seed disease-resistant treatment method |
| CN104894018A (en) * | 2015-05-27 | 2015-09-09 | 中国热带农业科学院热带生物技术研究所 | Luna Lin Haresii streptomycete and application thereof |
| CN104894018B (en) * | 2015-05-27 | 2018-09-25 | 中国热带农业科学院热带生物技术研究所 | A kind of Lu Nalinrui streptomycetes and its application |
| CN106244491A (en) * | 2016-08-29 | 2016-12-21 | 佛山市艳晖生物科技有限公司 | A kind of banana stem leaves residuum decomposing microbial inoculum suppressing droop and preparation method thereof |
| CN107904196A (en) * | 2017-12-26 | 2018-04-13 | 中国热带农业科学院热带生物技术研究所 | A kind of Yan Shi streptomycetes and its application |
| CN107904196B (en) * | 2017-12-26 | 2020-04-07 | 中国热带农业科学院热带生物技术研究所 | Streptomyces yanshi and application thereof |
| CN108977381A (en) * | 2018-08-07 | 2018-12-11 | 广西壮族自治区农业科学院 | Raw bacillus amyloliquefaciens and its application in a kind of banana |
| CN110066755A (en) * | 2019-05-20 | 2019-07-30 | 江苏师范大学 | One plant of fructus arctii endogenetic streptomycete, the microbial bacterial agent containing the endophyte and application |
| CN110066755B (en) * | 2019-05-20 | 2020-10-27 | 江苏师范大学 | Burdock endophytic streptomycete, microbial agent containing endophytic streptomycete and application |
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| CN100360660C (en) | 2008-01-09 |
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